Many bacterial pathogens secrete virulence factors to perform a variety of biological functions, including formation of cytolytic pores, modulation of host immune responses, or scavenging nutrients. The Type 1 secretion system (T1SS) is widespread among pathogens and translocates toxins across the cell envelope in Gram-negatives. One T1SS example is found in hemorrhagic E. coli strains that secrete hemolysin, a pore forming toxin. T1SS consist of inner and outer membrane components. Integrated into and anchored to the innermembraneisanABCtransporterandaperiplasmicmembranefusionprotein,respectively,ofwhichthe ABC transporter energizes protein translocation. These components partner with an outer membrane porin to facilitate translocation across the outer membrane. T1SS substrates can be extremely long, ranging in size from 40 to >900kDa. Despite their lengths, the polypeptides are directly channeled from the cytosol to the exteriorofthecell.WealreadysolvedthecrystalstructureoftheT1SSABCtransporterPrtDinarestingstate. Because PrtD is only functional in a fully assembled T1SS and to gain insights into the mechanism of T1SS- protein secretion, we propose to determine the structure of a stalled T1SS translocation intermediate by cryo electron microscopy (Aim 1). Substrate translocation will be stalled by fusing a stably folded domain to the substrate?sNterminus.T1SStranslocatesubstratesCterminusfirst,whichformsastableCa2+bindingdomain intheextracellularmilieu.Thus,byintroducingastablyfoldeddomaintothesubstrate?sNterminus,theT1SS is trapped between folded cytosolic and extracellular domains. To generate these T1SS translocation intermediates,weassembledafunctionalT1SSinvivofromheterologouslyexpressedandindividuallytagged components. This system secretes the substrate PrtG into the culture medium and allows trapping of translocationintermediateswithGFP-fusedsubstrates. Another defense mechanism employed by many Gram-negative pathogens is the modification of lipopolysaccharides(LPS)withcomplexcarbohydrates(O-antigens),therebypreventingcomplement-mediated killing.O-antigensaremostlylinearpolysaccharidesabout100to400sugarunitslong.Acommonbiosynthetic pathwayinvolvesthesynthesisoffullyassembledandlipid-anchoredO-antigensonthecytosolicleafletofthe inner membrane, after which the polymer is transported to the periplasmic side by a specific ABC transporter beforebeingattachedtotheoutercoreoligosaccharideofLPS.Wealreadydeterminedthecrystalstructureof the O-antigen-translocating ABC transporter in an open conformation, revealing a continuoustransmembrane channelsuitabletoaccommodateapolysaccharidechain.Tounravelthemechanismbywhichthispolymeris translocation,weproposetodeterminethetransporter?ssubstrate-boundstructureusingO-antigensofdefined lengths, synthesized in vivo or in vitro (Aim 2). Substrate-bound complexes will be generated by co- crystallizationorcrystal-soakingexperimentswiththepurifiedsubstrates.